Human marrow stromal cell lines which sustain hematopoiesis

ABSTRACT

Immortalized human stromal cell lines sustain and expand human hematopoietic precursor cells. The precursor cells are obtained from a blood product and inoculated into a culture medium conditioned by exposure to a human stromal cell line. Preferred human stromal cell lines secrete SCF, LIF, MIP1α, and IL-6, as exemplified by a human stromal cell line designated HS-1. The conditioned culture medium may be supplemented with additional growth factors, such as interleukin-3. After expansion the human hematopoietic precursor cells are harvested and returned to a patient or frozen and stored. The immortalized human stromal cell lines can also be used as feeder layers in ex vivo bone marrow cultures or in colony forming assays.

GOVERNMENT SUPPORT

The present invention was made with support under grant no. DK34431received from the U.S. National Institutes of Health. The U.S.Government has certain rights in this invention.

This is a Division of application Ser. No. 08/277,883 filed Jul. 20,1994 now abandoned.

BACKGROUND OF THE INVENTION

Hematopoietic cells are believed to arise in the bone marrow from atotipotent stem cell. The stem cell is able to renew itself as well asto give rise to progenitor cells such as the erythroid progenitors andmyeloid progenitors. The progenitor cells, in turn, give rise todifferentiated cells which are morphologically recognizable as belongingto a certain lineage such as the erythroid, megakaryocytic, myeloid, andlymphoid lineages, and which have a limited or no capacity toproliferate. In humans, stem cells and progenitor cells express the CD34antigen, while more differentiated hematopoietic cells do not.

Stem cells and progenitor cells do not execute their developmentprograms autonomously. Activities produced in the marrowmicroenvironment signal the progenitor cells to divide anddifferentiate. Thus, defining the functional components of the bonemarrow microenvironment is a prerequisite to understanding how theproliferation and differentiation of progenitor cells is coordinatelyregulated. The cellular complexity of the marrow microenvironment hasbeen demonstrated both in situ and in vitro by a variety ofhistochemical techniques (Lichtman, Exp. Hematol. 9:391 (1981), andAllen et al., Exp. Hematol. 12:517 (1984)). The marrow microenvironmentis comprised of both hematopoietic and stromal or mesenchymal derivedcells. The stromal cells include endothelial cells that form the sinusesand adventitial reticular cells that have characteristics consistentwith adipocytes, fibroblasts, and muscle cells (Charbord et al., Blood66:1138 (1985), and Charbord et al., Exp. Hematol. 18:276 (1990)).Numerous advances in recent years have provided considerable informationon the ontogeny and development of hematopoietic cells; however,ontogeny of the stromal components and their precise role in controllinghematopoiesis has proven elusive (Ogawa, Blood 81:2844 (1993);Muller-Sieburg et al., Critical Rev. Immunol. 13:115 (1993); andDorshkind, Ann. Rev. Immunol. 8:111 (1990)).

Long term cultures of marrow cells are an in vitro approximation of thein vivo marrow microenvironment and have been informative with respectto the identification of growth factors, adhesion proteins andextracellular matrix proteins that mediate the interaction between thehematopoietic cells and the stromal elements (Muller-Sieburg et al.,supra; Dorshkind, supra; Liesveld et al., Exp. Hematol. 9:391 (1981);Kittler et al., Blood 79:3168 (1992); Eaves et al., Blood 78:110 (1991);Clark et al., Bailliere's Clin. Haematol. 5:619 (1992); and Heinrich etal., Blood 82:771 (1993)). One improvement to this system was the use ofstromal precursors, positive for the STRO-1 antigen, to initiate longterm cultures (LTC); STRO-1 positive stromal precursors are devoid ofmyeloid components and less heterogeneous than primary cultures, but arestill capable of supporting hematopoiesis (Simmons and Torok-Storb,Blood 78:55-62 (1991)). However, both the STRO-1 initiated cultures andthe primary LTC are too complex to delineate contributions fromindividual cell types. Moreover, primary cultures can be highly variableand change with time, further complicating the identification of stromalcells that have a role in controlling hematopoiesis.

Immortalized stromal cell lines have been used to circumvent some ofthese problems. Numerous spontaneous murine cell lines have beenestablished (Zipori et al., J. Cell Physiol. 118:143 (1984); Zipori etal., J. Cell Physiol. 122:81 (1985); and Song et al., Exp. Hematol.12:523 (1984)), however, unlike mouse lines human cell lines undergosenescence unless first immortalized by transformation with a retrovirus(Lanotte et al., J. Cell Sci. 50:281 (1981)). The few human bone marrowstromal cell lines that are available were established using the SV40virus large T antigen (Harigaya et al., Proc. Natl. Acad. Sci. USA82:3477 (1985); Tsai et al., J. Cell Physiol. 127:137 (1986); Novotny etal., Exp. Hematol. 18:775 (1990); Slack et al., Blood 75:2319 (1990);Singer et al., Blood 70:464 (1987); Cicutinni et al., Blood 80:102(1992); and Thalmeir et al., Blood 83:1799 (1994)). Some of these linesare promising with respect to the maintenance of hematopoietic cells;unfortunately, some also display transformed phenotypes which limitstheir usefulness for extrapolation to the normal marrow microenvironment(Novotony, supra).

The ability to culture hematopoietic cells and their precursors, derivedfrom the bone marrow, peripheral blood, or umbilical cord blood of apatient or donor, offers the potential to overcome the disadvantages ofimmunosuppressive or immunodestructive therapies which are often used inthe treatment of cancer and other life-threatening diseases. Culturedhematopoietic cells can be used as an important source of proliferatingcells to reconstitute a patient's blood-clotting and infection-fightingfunctions subsequent to therapy. In addition, the ability to expandhematopoietic cells and their precursors in vitro may relieve dependenceon bone marrow aspiration or multiple aphereses as the only means ofobtaining sufficient cells for transplantation.

Early work in the field of hematopoietic stem cell culture centeredaround the culture of murine bone marrow aspirates in agar gel or liquidmedium. Unfractionated bone marrow (including stem cells, progenitorcells, more differentiated hematopoietic cells, and stromal elements)was used to inoculate the cultures, but they were generally short-livedand resulted in little or no increase in cell number, particularly inthe stem cell and progenitor compartments. The results were even lesspromising when human bone marrow was employed. The human cells generallyadhered to the bottom and sides of the culture vessel and their removalwas difficult.

Subsequent efforts focused on inoculating mouse bone marrow ontopreestablished monolayers of bone marrow stromal cells (so-called Dextercultures; Dexter, Acta Haematol, 62:299-305, 1979). While some successwas obtained with Dexter cultures of mouse cells, the same approach wasdisappointing with human cells, in that a steady decline in the numbersof all cell types is observed in human Dexter cultures (Quesenberry,Curr. Topics Microbiol. Immunol. 177:151 (1992)).

A further disadvantage of Dexter cultures is that, to the extent thatthere is expansion of hematopoietic precursor cells, these cells adhereto the stromal layer and are extremely difficult to recover from theculture without employing conditions which damage the cells. Theproliferating cells which are released into the culture medium (that is,the non-adherent cells) are generally more mature cells, which cannotrestore sustained hematopoiesis in a transplanted individual.

There remains a need in the art for a method of culturing humanhematopoietic cells, which method (a) results in expansion of the numberof hematopoietic precursor cells; (b) enhances the yield and recovery ofthe precursor cells without compromising viability; and (c) can beindependent of the presence of bone marrow stromal elements. Quitesurprisingly, the present invention fulfills this and other relatedneeds.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for sustainingand/or expanding the number of human hematopoietic precursor cells. Inone embodiment the method for sustaining or expanding the humanhematopoietic precursor cells includes inoculating the cells from ablood product, such as bone marrow, umbilical cord blood, or peripheralblood, into a culture vessel which contains a culture medium that hasbeen conditioned by exposure to a human stromal cell line. A preferredhuman stromal cell line secretes at least LIF, KL, MIP1α, and IL-6, andis exemplified by the human stromal cell line designated HS-5. Theconditioned culture medium of the invention may be supplemented with atleast one exogenously added growth factor, such as, for example,granulocyte colony stimulating factor, stem cell factor, interleukin-3,PIXY-321 (GM-CSF/IL-3 fusion), etc. The hematopoietic precursor cellsare optionally separated from mature hematopoietic cells presentinitially in the blood product prior to inoculating the conditionedculture medium. Further, the separated hematopoietic precursor cells maybe frozen initially for storage, and then thawed prior to inoculatingthe conditioned medium. Typically the cells will be cultured for a timeand under conditions sufficient to achieve at least an approximatelytwo- to five-fold expansion in the number of precursor cells relative tothe number of cells present initially in the blood product. After thedesired expansion or maintenance has taken place, the humanhematopoietic precursor cells can then be harvested from the culturemedium and returned to a patient, or frozen and stored.

In other aspects the invention provides compositions for sustaining orexpanding the number of human hematopoietic precursor cells. In oneembodiment the composition comprises a nutrient medium that has beenconditioned by exposure to an immortalized human stromal cell line, suchas the HS-5 line. The composition may also be supplemented with at leastone exogenously supplied growth factor, such as granulocyte colonystimulating factor, stem cell factor, interleukin-3 or PIXY-321, etc. Inother embodiments the invention provides an immortalized human stromalcell line which sustains the proliferation of human hematopoieticprecursor cells. Preferred lines produce cytokines such as LIF, KL,MIP1α and IL-6, as exemplified by the preferred line, HS-5.

The immortalized human stromal cell lines of the invention can also beused as feeder layers in ex vivo bone arrow cultures or in colonyforming assays, such as the methylcellulose assay for CFU-GM.Alternatively, the cell lines of the instant invention may be used tocondition medium, which medium may then be used to sustain and/or expandex vivo cultures of human hematopoietic precursor cells, or to sustaincolony forming assays. In a further aspect of the invention, mediumconditioned by exposure to the immortalized human stromal cell lines mayalso be used in vivo to promote hematopoiesis in patients whose bonemarrow function is compromised.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of two experiments that represent the range ofCFC production by primary LTC and the stromal cell lines HS-5, 23, 27 inFIG. 1A and HS-5, 21, and 27 in FIG. 1B. N=3 for FIG. 1A, and N=4 forFIG. 1B. Data are reported as the mean of the absolute number of CFCproduced from adherent and non-adherent layers in replicate culturesthat were initiated with 1500 38^(lo) cells. Error bars represent S.E.M.

FIG. 2 shows the small-scale expansion of 38⁺ cells with growth factormix (FIG. 2A), HS-5 conditioned medium (FIG. 2B), and HS-21 conditionedmedium (FIG. 2C). The same number of cells were added per well at timezero, expanded with different media for 5 days and stained with ethidiumbromide and acridine orange.

FIG. 3 shows the number of hematopoietic colonies grown from 38⁺ or38^(lo) cells in the presence of GF mix, HS-5 conditioned medium, HS-21conditioned medium with serum (s) or serum deprived (sd). FIG. 3A showsgranulocytic/monocytic colony numbers (G/GM) and FIG. 3B shows erythroidbursts (BFU-E). RPMIs represents RPMI media supplemented with 10% FCS.Results significantly different from HS-5 are designated 1, and resultssignificantly different from GF mix are designated 2. Error barsrepresent S.E.M., "*" indicates P<0.01, and "**" indicates P<0.05.

FIGS. 4A and B collectively depict the ELISA results demonstrating thesimilarity in cytokines secreted by HS-5 (solid bars) and HS-21 (openbars). "<std." indicates that the cytokine level was below thedetectable limits of the ELISA, and ">std." indicates that the level wasgreater than the standard curve. The supernatants were analyzed neat, at1:2 and 1:5 dilutions. Data represents the concentration from one ormore dilutions that were within the standard curve.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides compositions and methods for increasingthe number of human hematopoietic precursor cells in vitro and in vivo.Specifically, the present invention provides immortalized human stromalcell lines that can be used as feeder layers to sustain the growth anddifferentiation of human hematopoietic precursor cells ex vivo. Inanother aspect, the immortalized human stromal cell lines of the presentinvention can be used to condition medium, which medium can be used inaddition to or in lieu of a feeder cell layer and/or exogenously addedgrowth factors to support the growth of human hematopoietic precursorcells.

Human hematopoietic precursor cells are separated from a blood product,such as bone marrow, peripheral blood, or umbilical cord blood of apatient or donor, fetal peripheral blood and other sources. As discussedin more detail below, such separation may be performed, for example, byimmunoselection on the basis of their expression of an antigen, such asthe CD34 antigen, which is present on substantially all hematopoieticprecursor cells, but is substantially absent from more maturehematopoietic cells. The separated hematopoietic precursor cells may bestored frozen and thawed at a later date for inoculation into a suitablevessel containing a culture medium comprising a conditioned medium andnutritive medium, optionally supplemented with a source of growthfactors and, optionally, human or other animal plasma or serum.Alternatively, the separated cells may be inoculated directly intoculture without first freezing. In both cases the resultant cellsuspension is cultured under conditions and for a time sufficient toincrease the number of hematopoietic precursor cells relative to thenumber of such cells present initially in the blood product. The cellsmay then be separated by any of a variety of methods, such ascentrifugation or filtration, from the medium in which they have beencultured, and may be washed one or more times with fresh medium orbuffer. Optionally, the cells may be re-separated into CD34-positive and-negative fractions, prior to resuspension to a desired concentration ina medium or buffer suitable for infusion. The cells may then be infusedinto a patient or stored frozen for infusion at a later date.

Surprisingly, separated precursor cells, such as CD34-positive cells,will expand in number when cultured in the presence of conditionedmedium containing expressed products of bone marrow stromal elements,enabling clinically practicable expansion and recovery of hematopoieticprecursor cells. By working with separated precursor cells, the volumesof cells and culture fluids which must be handled are reduced to moremanageable numbers. Further, a high degree of expansion can be achievedwhen one starts with separated CD34-positive cells, rather than with anunseparated blood product. This is believed to be due to the removal ofcells otherwise present in the blood product, which inhibit expansion ofthe precursor cells. Under the conditions employed in the methods ofthis invention, cell recovery is greatly facilitated and viability ispreserved. Most importantly, the yield of hematopoietic precursor cells,capable of mediating both long-term and short-term hematopoieticrecovery in a myelosuppressed or myeloablated host, is increased. Theability to sustain or expand hematopoietic precursor cells in vitro orin vivo by the compositions and methods of the present invention isexpected to have tremendously important consequences for diseasetreatments which are inherently myelosuppressive or myeloablative, suchas in cancer chemotherapy.

Within the context of the present invention, hematopoietic precursorcells include those cells which express the CD34 antigen, among othersurface antigens, and include totipotent stem cells as well as committedprogenitor cells. The level of expression of the CD34 antigen will varyfrom one cell type to another. Consequently, a cell is operationallydefined as CD34-positive if it expresses sufficient CD34 antigen to bedetected by a given method of assay. For example, CD34-positive cellscan be identified by flow microfluorimetry using afluorescence-activated cell sorter (FACS), by immunofluorescence orimmunoperoxidase staining using a fluorescence or light microscope, byradioimmunoassay, or by immunoaffinity chromatography, among numerousother methods which will be readily apparent to one skilled in the art(see, for example, Lansdorp and Thomas (in Bone Marrow Processing andPurging, A. P. Gee (ed.), Boca Raton: CRC Press (1991) pg. 351).Hematopoietic precursor cells can also be detected by variouscolony-forming assays, such as CFU-GM and CFU-S assays (see, e.g.,Sutherland et al., in Bone Marrow Processing and Purging, supra at p.155).

Hematopoietic precursor cells, including CD34-positive cells, may beobtained from any of a variety of blood products, including bone marrow,peripheral blood, umbilical cord blood, fetal liver, and spleen. Bonemarrow is a particularly rich source of precursor cells (1-2% ofmarrow), but alternate sources may be preferable because of thediscomfort associated with bone marrow aspiration. Bone marrow istypically aspirated from the iliac crest, but may be obtained from othersites (such as the sternum or vertebral bodies) if necessitated by prioror concurrent disease or therapy.

Peripheral blood contains fewer precursor cells (typically <1% ofperipheral blood mononuclear cells), but is generally easier to obtainthan bone marrow. The number of precursor cells circulating inperipheral blood can be increased by prior exposure of the donor tocertain growth factors, such as, for example, G-CSF or SCF (KL), and/orcertain drugs, such as, for example, 5-fluorouracil, cyclophosphamide orprednisone (Korbling and Martin, Plasma Ther. Transfer Technol. 9:119(1980)). Peripheral blood collected from patients or donors who havebeen pretreated to increase the number of circulating CD34-positivecells is referred to as having been "mobilized." Depending upon thevolume which is desired, blood may be obtained by venipuncture or by oneor more aphereses, for example, on a COBE 2997 blood separator.Precursor cells can also be obtained from umbilical cord blood at thetime of delivery, either by simple gravity-induced drainage or manualexpression as described in U.S. Pat. No. 5,004,681, incorporated hereinby reference.

Although one can readily separate a bone marrow or peripheral bloodspecimen or apheresis product into precursor and mature cells, (such asCD34-positive and CD34-negative populations), it is generally preferredto prepare a buffy coat or mononuclear cell fraction from thesespecimens first, prior to separation into the respective populations.Methods for the preparation of buffy coats and mononuclear cellfractions are well-known in the art (Kumar and Lykke, Pathology 16:53(1984)).

Separation of precursor cells from more mature cells can be accomplishedby any of a variety of methods known to those skilled in the art,including immunoaffinity chromatography (Basch et al., J. Immunol.Methods 56:269 (1983)), fluorescence-activated cell sorting, panning(Wysocki and Sato, Proc. Natl. Acad. Sci. USA 15:2844 (1978)),magnetic-activated cell sorting (Miltenyi et al., Cytometry 11:231(1990)), and cytolysis. Generally, separation of a heterogeneouspopulation of cells, such as in a bone marrow aspirate or a peripheralblood specimen or apheresis product, into target (such as,CD34-positive) and non-target (such as, CD34-negative) fractions israrely complete. For the purposes of the present invention, separationis considered to have been accomplished if the target fraction iscomprised of at least about 20% precursor cells, more often about 50%precursor cells, and preferably about 70% precursor cells. In addition,it may be desirable to keep the total numbers of mature hematopoieticcells, such as platelets, granulocytes, and red cells, as low aspossible in order to prevent clumping and the release of degradativeenzymes which can adversely affect the recovery and viability ofengrafting cells, especially after freezing and thawing. Morespecifically, it may be desirable that the target fraction be comprisedof less than about 5% platelets, 50% granulocytes, and 10% red cellsand, preferably, less than about 1% platelets, 25% granulocytes, and 1%red cells.

Precursor cells may be positively selected or negatively selected. Bypositive selection is meant the capture of cells by some means, usuallyimmunological, on the basis of their expression of a specificcharacteristic or set of characteristics (usually an antigen(s)expressed at the cell surface). For example, CD34-positive cells can bepositively selected by any of the above methods (except cytolysis, whichwould result in destruction of the desired cells) on the basis of theirexpression of the CD34 antigen utilizing an anti-CD34 antibody, such asthe monoclonal antibodies 12.8, My-10, and 8G12 (commercially availablefrom Becton Dickinson Co., Mountain View, Calif.), or Q-Bend 10(commercially available from Biosystems Ltd., Waterbeach, Cambridge,England).

Negative selection means the exclusion or depletion of cells by somemeans, usually immunological, on the basis of their lack of expressionof a specific characteristic or set of characteristics (again, usually asurface antigen). For example, CD34-positive cells can be negativelyselected by any of the above methods on the basis of their lack ofexpression of lineage-defining antigens, such as CD 19 (for Blymphocytes), CD3 (for T lymphocytes), CD56 (for NK cells), etc.,utilizing antibodies to the above-mentioned and other lineage-definingantigens. By using a cocktail or mixture of monoclonal antibodiesdirected to red cell, platelet, granulocyte, lymphocyte and/or tumorcell antigens, it is possible to leave behind a population of cellswhich is highly enriched for CD34-positive cells. Numerous monoclonaland polyclonal antibodies suitable for this purpose are known in the art(see Leukocyte Typing IV, Knopp et al. (eds.), Oxford UP, 1989) and arecommercially available from a wide variety of sources (for example,Becton Dickinson Co., Mountain View, Calif.; Coulter Immunology,Hialeah, Fla.; Ortho Diagnostics, Raritan, N.J., etc.).

Alternatively, precursor cells can be separated from mature cells by acombination of negative and positive selection techniques. A preferredcombination of negative and positive selection techniques is comprisedof a first selection for CD34-positive cells utilizing an anti-CD34antibody, followed by a second selection forHLA-DR-negative/CD34-positive cells, using an anti-HLA-DR antibody to anon-polymorphic determinant on the DR molecule. Antibodies tonon-polymorphic determinants on the HLA-DR molecules are well-known inthe literature (see Knopp et al., supra) and are available from avariety of sources, including those mentioned above. An example of asuitable monoclonal anti-HLA-DR antibody is the antibody produced by thehybrid cell line L243 (Lampson et al., J. Immunol. 125:293 (1980)),which cell line is available from the American Type Culture Collection(Rockville, Md.) under the designation ATCC HB55. The advantage of thisor other dual selection strategies is that the volume of cells which isplaced into culture is smaller and thus more manageable.

Although selection of CD34-positive cells usually involves the use ofone or more antibodies or fragments thereof, in some cases selection mayinvolve the use of lectins or other types of receptors or ligandsexpressed on the cell surface. Among other antibodies, antigens,receptors and ligands which may be useful, alone or in combination withother markers, for separating CD34-positive cells from CD34-negativecells are transferrin, the transferrin receptor, soybean agglutinin,c-kit ligand, c-kit receptor, HLA-DR, CD33, etc.

Within another aspect of the invention, the precursor cells areperiodically separated from more mature cells. Briefly, mature cells(which include not only terminally differentiated blood cells, but cellsof an intermediate lineage) may inhibit the expansion anddifferentiation of precursor cells via a feedback control mechanism.Removal of more mature cells from a culture thus permits expansion ofthe precursor cells to many times their original numbers. Within thecontext of the present invention, "periodically separating" meansremoval of mature cells at least every 7 days, preferably every 4 days.

Various methods may be utilized in order to periodically separateprecursor from mature cells. For example, cells can be separated on anaffinity column, incubated in a selected medium, and then subsequentlyreseparated in order to separate the precursor cells from the newlydifferentiated mature cells. Particularly preferred methods and devicesfor the selection of precursor cells, such as CD34-positive cells, aredescribed in U.S. Pat. Nos. 5,215,927, 5,225,353, 5,262,334 and5,240,856, each of which is incorporated herein by reference in itsentirety. These applications describe methods and devices for isolatingor separating target cells, such as hematopoietic precursor cells, froma mixture of non-target and target cells, wherein the target cells arelabeled, directly or indirectly, with a biotinylated antibody to atarget cell surface antigen. Labeled cells are separated from unlabeledcells by, flowing them through a bed of immobilized avidin, the labeledcells binding to the avidin by virtue of the biotinylated antibody boundto their surface, while the unlabeled cells pass through the bed. Afterwashing the bed material, the labeled (bound) cells can be eluted fromthe bed, for example, by mechanical agitation. A cell separator deviceis also provided for separating target cells from non-target cells,comprising (a) a column assembly which includes a column, a sample fluidsupply bag and a fluid collection bag wherein the column is provided forreceiving the sample fluid from the sample fluid supply bag and forseparating the target cells from the sample fluid and retaining thetarget cells, and wherein the fluid collection bag is provided forreceiving the target cells after being released from the column, (b) anagitation means for agitating the contents of the column to assist inreleasing the sample cells retained in the column, the agitation meansbeing responsive to a drive signal for varying amounts of agitation ofthe contents of the column to vary the rate at which the sample cellsare released, (c) a column sensor means for providing a column signalindicative of the optical density of fluid flowing out of the column andinto the fluid collection bag, (d) a column valve means responsive to acolumn valve control signal for selectively enabling the fluid comingout of the column to flow into the fluid collection bag, and (e) a dataprocessor means for controlling the operation of the cell separator, thedata processor means being responsive to the column signal for providingthe drive signal and the column valve control signal to preventinadequate concentrations of the target cells from being collected. Oneembodiment of this invention is the CEPRATE SC™ cell separation systemdescribed in Berenson et al. (Adv.

Bone Marrow Purging & Processings, N.Y.: Wiley-Liss, 1992, pg. 449).

Subsequent to separation, precursor cells are inoculated into a culturemedium comprised of a nutritive medium, any number of which, such asRPMI, TC 199, Ex Vivo-10, or Iscove's DMEM, along with a source ofgrowth factors, will be apparent to one skilled in the art.Proliferation and differentiation of precursor cells may be enhanced bythe addition of various components to the medium, including a source ofplasma or serum. Among sources of plasma or serum are fetal bovine andhuman. Particularly preferred are human autologous plasma or human AB⁻plasma which have been screened in accordance with standard blood bankprocedures to ensure the absence of infectious agents, such as HBV orHIV. The amount of plasma or serum which is used will vary, but isusually between about 1 and 50% (by volume) of the medium in which thecells are grown, and more often between about 1 and 25%.

According to one aspect of the present invention, separated precursorcells are cultured in a nutritive medium containing a source of plasmaor serum, which medium has been previously conditioned by exposure toimmortalized stromal cells for a variable period of time and underconditions sufficient to allow those cells to secrete products, such asgrowth factors, into the medium. For example, conditioned mediumsuitable for the culture of separated CD34-positive cells may beprepared by inoculating an immortalized stromal cell line HS-5 asdescribed herein into a nutrient medium (optionally containing plasma orserum), allowing the cells to grow, usually for 1 to 3 days, and thenseparating the cells from the medium (for example, by centrifugation orfiltration). Optionally, the conditioned medium may be sterilized and/orconcentrated prior to use and/or supplemented by the addition ofexogenous growth factors.

Although the HS-5 stromal cell line is particularly preferred forgenerating conditioned medium, other cell lines can be prepared andselected according to the present invention which secrete a variety ofgrowth factors and which may be used to prepare the conditioned mediumfor short term or long term support of hematopoiesis. Typically, suchcell lines are prepared by transfecting a long term marrow culture witha retroviral supernatant, the retrovirus carrying an oncogene,integration of which leads to immortalization of the transfected celland its progeny. The retroviral vector may also carry a gene for aselectable marker, such as neomycin resistance, to facilitateidentification of transfected cells. Following transfection, cells arecloned and characterized morphologically and histochemically, as well asfunctionally to ascertain their ability to sustain hematopoiesis exvivo. Growth factors expressed by the resultant cell lines can beassayed, for example, by ELISA or RIA.

In addition, it will be apparent that in some instances it may bedesirable to inoculate multiple cell lines simultaneously to producemedium conditioned by more than one line. Alternatively, differentbatches of medium can be conditioned by different cell lines and thebatches combined, after the cells have been separated and discarded, toachieve the same effect.

The length of time for which medium is conditioned may vary from 1 dayto 2 weeks, but will usually be between 1 day and 1 week and more often,between 1 day and 5 days. In addition to conditioning the medium byexposing it to immortalized stromal cells such as the HS-5 cell line,the medium may also be supplemented by the addition of one or morepurified or partially purified growth factors, such as those mentionedabove. The term "conditioned medium" is used to include mediumconditioned solely by exposure to cells as well as medium conditioned byexposure to cells and supplemented with exogenous growth factors.

Conditioned medium may be prepared with or without a source of serum orplasma. If used, the serum or plasma may be of human or other animalorigin. Particularly preferred is human autologous plasma or human AB⁻plasma which has been screened in accordance with standard blood bankprocedures to ensure the absence of infectious agents. The amount ofplasma or serum which is used will vary, but is usually between about 1and 50% (by volume) of the medium in which the cells are grown, and moreoften between about 1 and 25%.

The conditioned medium of the present invention may be concentratedprior to use by a variety of means, for example, by ultrafiltration,although other concentrating means will also suffice. The amount ofconcentration will vary, but is usually between 2 and 100-fold, moreoften between 2 and 50-fold, and most often between 2 and 10-fold.Separated precursor cells may be inoculated directly into conditionedmedium (concentrated or non-concentrated) or they may be inoculated intoa mixture of conditioned (concentrated or non-concentrated) andnon-conditioned medium (with or without exogenously supplied growthfactors and serum or plasma). If inoculated into a mixture ofconditioned and non-conditioned medium, the ratio of conditioned(non-concentrated) to nonconditioned medium will usually be between 1:1and 1:10 (on a volume basis), more often between 1:1 and 1:5, and mostoften between 1:1 and 1:2. Although these ratios are expressed fornon-concentrated conditioned medium, it will be apparent to thoseskilled in the art that the equivalent ratios can be obtained usingsmaller volumes of concentrated conditioned medium.

Among growth factors which may be advantageously employed in the mediumare interleukins (IL) 1-15, erythropoietin (EPO; U.S. Pat. No.4,703,008, incorporated herein by reference), stem cell factor (SCF,also known as mast cell growth factor and c-kit ligand), granulocytecolony stimulating factor (G-CSF), granulocyte, macrophage-colonystimulating factor (GM-CSF), macrophage-colony stimulating factor(M-CSF), transforming growth factor beta (TGF beta), tumor necrosisfactor alpha (TNF alpha), the interferons (IFN alpha, beta, or gamma),fibroblast growth factor (FGF), platelet-derived growth factor (PDGF),insulin-like growth factors (IGF-1 and IGF-2), megakaryocyte promotingligand (MPL) and SLK-2, etc. Growth factors are commercially available,for example, from R&D Systems (Minneapolis, Minn.). Particularlypreferred are combinations of growth factors, especially the combinationof SCF, IL-1 alpha, IL-3 (EP Publ. EP 275,598 and 282,185, incorporatedherein by reference) and IL-6. It may also be desirable to selectivelyremove inhibitors of hematopoiesis, as described in, e.g., Maxwell etal., using an antibody, soluble receptor or the like.

In general, the above-mentioned growth factors are purified or partiallypurified before they are added to the culture medium. Usually, they willbe produced by recombinant DNA methods, but they may also be purified bystandard biochemical techniques from conditioned media.Non-naturally-occurring growth factors can also be produced byrecombinant DNA methods, for example, PIXY-321 is a fusion protein whichhas both GM-CSF and IL-3 activity, as described in U.S. Pat. No.5,108,910, incorporated herein by reference. It will be evident to thoseskilled in the art that other fusion proteins, combining multiple growthfactor activities, can be readily constructed, for example, fusionproteins combining SCF activity with that of other growth factors suchas IL-1, IL-3, IL-6, G-CSF, and/or GM-CSF.

The amount of each growth factor to be used is determined empiricallyand will vary depending on the purity and method of production of thefactors. Generally, concentrations between 0.5 and 100 ng/ml aresufficient, more often between 0.5 and 50 ng/ml. Where more than onegrowth factor is used, the optimum amount of each factor should bedetermined in combination with the other factors to be used. This isbecause some growth factors can modulate the activity of other growthfactors, necessitating that they be used sequentially rather thansimultaneously, while in other instances, growth factors may actsynergistically. Still other growth factors may enhance proliferation ordifferentiation along one pathway, while suppressing another pathway ofinterest.

Separated precursor cells may be cultured in any vessel which is capableof being sterilized, is adapted or adaptable to gas exchange with theatmosphere, and is constructed of a material which is non-toxic tocells. A variety of vessels suitable for this purpose are well-known inthe art, including stirring flasks (Corning, Inc., Corning, N.Y.),stirred tank reactors (Verax, Lebanon, N.H.), airlift reactors,suspension cell retention reactors, cell adsorption reactors, and cellentrapment reactors, petri dishes, multiwell plates, flasks, bags andhollow fiber devices. If agitation is desired, it can be attained by anyof a variety of means, including stirring, shaking, airlift, orend-over-end rotation. In addition to maintaining the culture insuspension by agitating the medium (as by stirring or airlift), theculture can also be maintained in suspension by matching the density ofthe culture medium to the density of the cells or microcarrier beads.

The immortalized human stromal cell lines of the instant invention canbe used as feeder layers in ex vivo bone marrow cultures or in colonyforming assays, such as the methylcellulose assay for CFU-GM or thecobblestone area forming cell (CAFC) assay. Alternatively, the celllines of the instant invention may be used to condition medium, whichmedium may then be used to sustain and/or expand ex vivo cultures ofhuman hematopoietic precursor cells, or to sustain colony formingassays, such as the CFU-GM and CAFC assays. For example, methylcelluloseassays are typically performed using conditioned medium from lymphocytesstimulated with the lectin phytohemagglutinin (PHA-LCM). Human stromalcell line conditioned medium (HS-CM) can be substituted for PHA-LCM inmethylcellulose assays. Medium conditioned by exposure to theimmortalized human stromal cell lines may also be used in vivo topromote hematopoiesis in patients whose bone marrow function iscompromised.

The following examples are offered by way of illustration, not by way oflimitation.

EXAMPLE I

Production of Human Stromal Cell Lines

This Example describes the production and characterization of HPV 16E6/E7 immortalized human marrow stromal cell clones. In subsequentExamples the stromal cell clones are shown to support the proliferationof hematopoietic progenitors and maintain colony forming cells (CFC) forup to 8 weeks in culture.

Adult bone marrow was obtained from normal donors and LTMC (long termmarrow cultures) were established as described by Gartner and Kaplan,Blood 56:117 (1980). Briefly, buffy coat cells from marrow aspirateswere plated in plastic tissue culture dishes at 1-2×10⁶ cells per ml.Adherent cells were grown in Long term culture (LTC) medium containingIscoves, 12.5% horse serum, 12.5% fetal calf serum, L-glutamine (0.4mg/mL), sodium pyruvate (1 mM), penicillin (100 U/mL), streptomycinsulfate (100 μg/mL), hydrocortisone sodium succinate (10⁻⁶ M) andβ-mercaptoethanol (10⁻⁴ M).

For immortalization of bone marrow cells lines, the LTMCs were infectedwith the amphitrophic LXSN-16 E6/E7 retrovirus that was packaged in thePA317 cell line as described in Halbert et al., J. Virol. 65:473 (1991),incorporated herein by reference. Primary LTMC were exposed to virus inthe presence of 4 μg/ml polybrene (Aldrich Chemical Co. Inc., Milwaukee,Wis.) for 2 hours at 37° C. The virus containing medium was removed andthe cells were incubated for an additional 5 hours with mediumcontaining polybrene. Cells were then washed and fed with LTC medium andincubated an additional 48 hours. Cell cultures were then trypsinizedand replated at limiting dilution. Transduced clones were selected with50 μg/ml G418 and resistant colonies were picked and grown in LTC mediumusing standard tissue culture techniques. Following expansion mostclones were switched to RPMI containing 10% serum and HS-5 was switchedto serum deprived medium containing 1% Nutridoma (Boehringer-Mannheim),100 mM glutamine, 100 mm sodium pyruvate, 100 U/mL penicillin-streptomycin in Iscoves media.

Twenty-seven foci were identified and isolated using cloning rings toestablish stromal cell lines (HS-1 to HS-27) of which twenty-four wereretained and proved to be resistant to G418 at 50 μg/ml. All lines wereinitially characterized morphologically and histochemically, screenedfor maintenance and/or proliferation of HPs (see below) and then frozen.Several clones designated HS-5, HS-21, HS-23 and HS-27 were selected formore detailed analysis and have been maintained in continuous culturefor up to 20 months with periodic analysis of their phenotypes.

Based on morphology two distinct cell types were observed, smallfibroblastic (HS-5, HS-21) and large flattened epithelioid (HS-23,HS-27). The two fibroblastic lines, although similar in morphology,differed in regard to growth patterns. HS-5 formed a reticulum ofoverlapping cells, reminiscent of astrocytes, whereas HS-21 cells werewell separated and lined up in parallel arrays. At higher densities HS-5formed a dense "net" of cells, whereas HS-21 formed a contiguousmonolayer with discernible cell boundaries. HS-23 and HS-27 formed largeflattened polygonal shaped cells that exemplify "blanket" cells andmaintain numerous intercellular contacts with neighboring cells. HS-23and -27 also formed monolayers, however because of their flattenedmorphology it was difficult to identify distinct cell boundaries.

Southern hybridization on genomic DNA from the 4 cell lines was usedfirst to confirm that LXSN-16 E6/E7 had integrated, and second toestablish clonality. Genomic DNA was isolated from 1×10⁷ stromal cellsusing a modification of the technique described in Ausebel et al.,(eds.) Current Protocols In Molecular Biology, New York, WileyInterscience (1987). Prior to southern hybridization 10 μg of genomicDNA was digested with excess EcoRI overnight at 37° C. The DNA wasextracted with phenol:chloroform and precipitated. The digested genomicDNA (10 μg) was separated on a 0.5% agarose gel in TBE and thentransferred to a nylon membrane according to manufacturersspecifications (Hybond, Amersham). The membrane was hybridized withrandom primed probes generated against the E6E7 insert. 50,000 cpm washybridized overnight at 42° C. and washed 2× with 2× SSPE at 25° C. andthen washed 2 more times with 0.2× SSPE containing 0.1% SDS at 60° C.prior to autoradiography. The autoradiographs indicated that all celllines contained retroviral insert(s) with only a single band present inHS-5 and HS-23. However HS-21 and HS-27 had two bands, indicating eitherthat they contained two inserts or that two clones contribute to theline. Analysis of foreskin fibroblasts and plasmid DNA indicated thatthe probe was specific for the LXSN 16 E6/E7 integrant.

The antigenic phenotypes of the stromal cell lines were then determinedby routine immunochemistry procedures using a variety of markers. Thefollowing antigens were identified with available monoclonal antibodies:Smooth muscle actin (monoclonal antibody IA4-IgG2a; Sigma); CD14(monoclonal antibody leuM3-IgG2b, Becton-Dickinson) was used as a markerfor macrophages; FVIII antigen (human type 1) was identified withmonoclonal antibody obtained from Calbiochem, La Jolla, Calif.) as amarker for endothelial cells; Monoclonal antibody 6.19-IgG2a was used toidentify fibroblasts, endothelial cells and adipocytes (obtained from C.Frantz, University of Rochester School of Medicine and Dentistry,Rochester N.Y.); CD34 was identified with monoclonal antibody 12.8 (IgM,I. Bernstein, Fred Hutchinson Cancer Research Center [FHCRC]);fibronectin and vimentin were identified with monoclonal antibody P1H11and P1H1-C9, respectively (obtained from W. Carter, FHCRC); Class I MHCantigen was identified with monoclonal antibody 60.5, (P. Martin,FHCRC); VLA-4 and VCAM-1 were identified with monoclonal antibodies (4B9ascites, J. Harlan, Univ. Washington); collagen Type I was identifiedwith MAB1340 (IgG), Type III was identified with MAB1343 (IgG1), andType IV was identified with MAB 1910 (IgG1), each obtained fromChemicon, Temecula Calif. Similar monoclonal antibodies can be readilyobtained and substituted for those used in this study to identify thecellular antigens of interest.

For immunofluorescence staining, semi-confluent cells were rinsed withwarm HBSS and fixed for 10 minutes with 1% formaldehyde in PBS at 25° C.The cells were washed with phosphate buffered saline (PBS) and treatedwith 0.2 M glycine in PBS for 5 minutes at 25° C. One additional washwas performed with PBS prior to incubation with a specific antibody orirrelevant non-specific isotype control antibody for 1 hr at 25° C.After incubation with the primary antibody the cells were washed 3× andincubated with a secondary antibody (goat anti-mouse IgG/IgM fluoresceinisothiocyanate (FITC)-conjugated antibody (Tago) for 1 hr at 25° C. andwashed with PBS prior to viewing with a Nikon Diaphot fluorescentmicroscope. Primary LTCs and foreskin fibroblasts (FSF) were used ascontrols for antibody staining.

The results of the indirect immunofluorescence, shown in Table 1,indicated that all cell lines were negative for MHC Class II (DR) andCD14, a macrophage specific marker, and positive for antigens normallyassociated with non-hematopoietic stromal cells. All lines expressedcollagen III and IV, with low levels of collagen I detected on HS-5 andHS-27. Analysis of VCAM-1 revealed that HS-5 and HS-21 expressed lowlevels, HS-23 was heterogeneously positive, and HS-27 was homogeneouslya strong positive.

                  TABLE 1                                                         ______________________________________                                                        Stromal Cell Lines                                                                  HS-                                                       Markers MoAB 5 HS-21 HS-23 HS-27                                            ______________________________________                                        Smooth Muscle Actin                                                                        IA4      +++    +++   +++   +++                                    MHC Class I 60.5 +++ ++ ++ ++                                                 MHC Class II P4.1 - - - -                                                     Fibroblasts, Adipocytes                                                       Endothelial cells 6.19 ++ ++ ++ ++                                            FVIII Agn                                                                     Macrophage (CD14) leuM3 - - - -                                               Endopeptidase (CD10) J5 +++ + + +                                             Fibronectin P1H11 ++ ++ ++ ++                                                 CD34 12.8 - - - -                                                             Stromal, Endothelial STRO-1 - - - -                                           Mesenchymal (Vimentin) P1H1-C9 + + + +                                        Collagen I MAB1340 + - - +                                                    Collagen III MAB1343 +++ + +++ ++                                             Collagen IV MAB1910 +++ + ++++ ++                                             VCAM-1 4B9 + + ++/- +++                                                       Alkaline Phosphatase  - +/- +/- +/-                                           Acid Phosphatase  +++ + +++ ++                                              ______________________________________                                    

Indirect-immunofluorescent and cytochemical analysis of the stromal celllines. +/- indicates that the cell lines were heterogeneously positiveand - represents the lack of detectable antigen. ++ indicates goodstaining and +++ indicates that the cells were strongly positive.

Cytochemical analysis for alkaline phosphatase activity was determinedusing cells fixed with 2% formaldehyde in absolute methanol for 30seconds at 4° C. (35), then washed with distilled water and air dried.After drying the cells were incubated at 37° C. for 30 minutes infiltered reaction buffer containing 0.2 M Tris HCl (pH 9.1), 1.0 mg/mLFastblue BB with or without 0.2 M napthal AS phosphate inN,N-dimethylformamide. After incubation the cells were washed withdistilled water, overlaid with Aqua Mount (Lerner Laboratories,Pittsburgh, Pa.) and photographed with a Nikon Diaphot microscope.

For analysis of acid phosphatase activity the cells were fixed with 60%acetone in 0.04 M citrate buffer (pH 5.4) for 30 seconds at 25° C.,rinsed with distilled water, air dried and incubated with the reactionbuffer. The reaction buffer was made up in 24 mls of 0.1 M acetatebuffer with or without 12.5 mg Napthal AS-BI phosphate as substrate and7.5 mg Fast Garnet GBC dye was added as counterstain. This solution wasfiltered through a Whatman #4 filter and then incubated with cells for 1hour at 25° C. protected from light. After staining the cells werewashed with distilled water, overlaid with Aqua-mount, photographed(Nikon Diaphot microscope) and scored for the presence of acidphosphatase.

As shown in Table 1, all lines were positive for acid phosphatase withsome differences in the degree of staining. In contrast, HS-5 wasnegative for alkaline phosphatase staining while all others wereheterogeneously positive.

The cell lines were also tested for their ability to undergo lipogenesisin response to corticosteroids. Confluent stromal lines were incubatedwith corticosteroids for 4 weeks and stained with oil red 0 to determineif these lines contain adipogenic cells as described in Kodama et al.,J. Cell Physiol. 112:83 (1982). Cultures were fed weekly with eitherdexamethasone (10⁻⁷ M), hydrocortisone (10⁻⁶ M), insulin (10 mg/Ml) ordexamethasone combined with insulin, in RPMI containing 10% FCS. Afterthe incubation period the cells were washed extensively with PBS andthen fixed with 10% formalin in PBS for 30 minutes. The excess formalinwas washed off with PBS and the cells were stained for 15 minutes withfiltered oil red 0 (0.3% w/v in isopropanol). The stain was thendifferentiated with 60% isopropanol, washed and the cells counterstainedwith Mayers hematoxylin for 30 seconds.

The results indicated that HS-5 and HS-21 cell lines did not accumulatelipids, whereas a few cells (approximately 1-2%) from HS-27 formed lipidvacuoles in the presence of all steroids tested. HS-23 formed lipidvacuoles in the presence of dexamethasone only. None of the lines,however acquired the large multilocular vacuoles commonly observed inadipocytes that are present in LTMCs (Gartner and Kaplan, supra, andEaves et al., J. Cult. Meth. 13:55 (1991).

Thus, these stromal cell lines have increased growth rates, do notundergo senescence (some have been in continuous culture for two years),and retain characteristics of normal differentiated bone marrow stromalcells. Positive staining with monoclonal antibody 6.19 (specific forfibroblasts, endothelial and adipocytes), P4.1 (CD10, endopeptidase),P1H11 (vimentin) and the absence of a macrophage marker (CD14) indicatesthat the cells are mesenchymal in origin. The lack of FVIII antigenindicates that they are not endothelial, however all lines expresscollagen type IV which is consistent with the endothelial nature of bonemarrow stroma (Novotny et al., Exp. Hematol. 18:775 (1990) and Zipori,in Handbook of the Hematopoietic Microenvironment, pp. 287-329, Ed. M.Tavassoli, Humana Press, Clifton N.J. 1989). Only the HS-23 cell lineresponded to dexamethasone, suggesting that it may be pre-adipocytic.The cell lines displayed a normal staining pattern for smooth muscleactin, vimentin, cell associated fibronectin and growth was inhibited atconfluency. CD34 and STRO-1 were absent, which is consistent with theloss of these markers in normal bone marrow cultures after several weeksof growth (Sutherland et al., Proc. Natl. Acad. Sci. USA 87:3584 (1990)and Simmons and Torok-Storb, Blood 78:55-62 (1991)). All 24 lines,except HS-5, were heterogeneously positive for both alkaline phosphataseand acid phosphatase.

Overall, the morphological and phenotypic characteristics of these celllines were similar to murine bone marrow stromal cell lines (Zipori etal., J. Cell Physiol. 118:143 (1984); Zipori et al., J. Cell Physiol.122:81 (1985); Song et al., Exp. Hematol. 12:523 (1984); and Zipori, inHandbook of the Hematopoietic Microenviroment, supra.), SV40 transformedhuman cell lines (Lanotte et al., J. Cell Sci. 50:281 (1981); Harigayaet al., Proc. Natl. Acad. Sci. USA 82:3477 (1985); Tsai et al., J. CellPhysiol. 127:137 (1986); Novotny et al., Exp. Hematol. 18:775 (1990);Slack et al., Blood 75:2319 (1990); Singer et al., Blood 70:464 (1987);Cicutinni et al., Blood 80:102 (1992); and Thalmeir et al., Blood83:1799 (1994)), and transient non-transformed human lines (Lanotte etal., supra). However, no spindle shaped cells were observed as othershave previously reported (Novotny et al., supra) and the HPVimmortalized lines remain MHC class II (DR) negative, as observed withnormal marrow (Novotony, supra).

EXAMPLE 2

Stromal Lines Support Short and Long Term Hematopoiesis

A rapid screening assay was developed to demonstrate the viability andexpansion of hematopoietic cells when co-cultured with the cell lines.

To isolate CD34+/38+ and 38lo cells, adult marrow was obtained fromcadaveric donors. The mononuclear cells were isolated by Ficoll densitycentrifugation and RBCs removed by hemolysis with 150 Mm NH₄ Cl₂ at 37°C. Marrow mononuclear cells were stored frozen in RPMI, 36% FCS, 10%DMSO, 90U Penicillin, 90 mg/ml streptomycin sulfate, and 0.36 mg/mlglutamine. The stored cells were thawed at 37° C. and slowly diluted onice to a final DMSO concentration below 1%. After washing, the CD34+cells were labeled with anti-CD34 conjugated to fluoresceinisothiocyanate (FITC) (HPCA-2 (IgG₁) Becton-Dickinson, San Jose Calif.)for 20 minutes on ice, washed with PBS containing 1% BSA and thenlabeled with rat anti-mouse IgG₁ conjugated to superparamagneticmicrobeads (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany) (Miltenyiet al., Cytometry 11:231 (1990). The CD34+ cells were positivelyselected using High-Gradient Magnetic Cell Sorting (Miltenyi BiotecGmbH). The CD34+ enriched population was then incubated with anti-CD38conjugated to phycoerythrin (PE) (leu-17, Becton-Dickinson) for 20minutes on ice, washed, and sorted using a FACStar Plus(Becton-Dickenson). Cells with medium to high forward light scatter andlow side scatter were selected and both the 38⁺ and the 38^(lo)population of CD34⁺ cells were collected (Reems and Torok-Storb, Blood,submitted (1994)).

Both 38^(lo) and 38⁺ cells were co-cultured on stromal cell lines inserum-deprived medium for five days with and without IL-3 (10 ng/Ml) andthen stained to differentiate stromal from hematopoietic cells andviable cells from dead cells. Screening was initiated by plating stromalcells at a density of 600 to a 1000 per well in Terasaki 96-well plates(Nunc) two days prior to addition of the bone marrow cells. The 38^(hi)and 38^(lo) cells (about 150-350 per well) were added to the cultures inserum deprived medium (Nutridoma-HU) and incubated at 37° C. in 5% CO₂humidified incubator for 5 days. The viability and proliferation ofprogenitors was scored after the addition of 5 μl of a staining mixturewhich contained 2.5% India ink, 250 μg/ml ethidium bromide and 75 μg/mlacridine orange in HBSS. The number of viable cells was determined foreach well by inverted fluorescence microscopy (Nikon Diaphotmicroscope).

All 24 cell lines maintained the viability of both 38⁺ and 38^(lo)subpopulations of CD34⁺ cells for 5 days. When IL-3 was added to theco-cultures the 38⁺ cells increased in number in all cases. However,cell line HS-5 was able to induce the 38⁺ cells to proliferate withoutexogenous IL-3, and the addition of IL-3 to HS-5 co-culture did notincrease the extent of proliferation beyond that observed with HS-5alone. Fluorescence microscopy revealed the differences between themaintenance of 38⁺ cells on HS-21 and the proliferation of these cellson HS-5. Small round cells were viable hematopoietic cells thataccumulated acridine orange and fluoresce green, whereas large flatcells were stromal cells, and nonviable cells incorporated ethidiumbromide and fluoresce orange. The same number of 38⁺ cells were platedinto each culture at the initiation of the experiment. No proliferationof 38^(lo) cells was observed within the 5 day time span of thisexperiment.

To determine if these stromal lines could support less maturehematopoietic cells their ability to maintain or produce colony formingcells (CFC) from the 38^(lo) population after 5 and 8 weeks wasdetermined. To demonstrate long-term support of CFC, two to four weekold primary LTMCs were established and maintained according to theguidelines of Gartner, supra, and Eaves, supra. Stromal cell lines wereirradiated at 2000 RADS and plated into 24 well plates at least 24 hoursprior to the addition of hematopoietic cells. Irradiated stromal cellswere plated at sufficient cell densities to ensure formation ofmonolayers. The stromal cultures were seeded with 1000-3000 38^(lo)cells and semidepleted weekly for 5 or 8 weeks. The non-adherent andadherent cells were harvested and analyzed for colony forming cellsusing methylcellulose colony assays.

Colony assays were performed using a stock solution of 1.2%methylcellulose, 2.5% BSA, 25% FCS (Hyclone 796), 100U penicillin, 100μg/ml streptomycin sulfate, and 0.1 M β-mercaptoethanol. Colonystimulating activity was provided either by a growth factor mixcontaining 10 ng/ml IL-1, IL-3, IL-6, G-CSF, GM-CSF, KL and 3U/ml EPO,or by 10% conditioned media (PHA-LCM). Hematopoietic cells (38⁺ or38^(lo)) plus 100 μl growth factor mix or conditioned media was added to0.9 mL of the methylcellulose stock. Colony formation was scored at day14 and designations were periodically confirmed by Wright-Giemsastaining of colony cytospins.

The two representative experiments are indicative of the range of CFCproduction and demonstrate that the stromal lines can maintain CFC atlevels comparable to primary LTMC for up to 8 weeks. HS-27, whichexpressed the highest levels of VCAM-1, was the only cell line toestablish cobblestone regions when incubated with 38^(lo) cells. Boththe fibroblastic and "blanket" cell lines supported CFC for 5 to 8 weeksat levels comparable to primary LTC. These results indicate thatindependent of phenotypic differences and detectable cytokine secretionthese diverse stromal cell lines produce hematopoietic maintenancefactors at levels that are sufficient to support immature pluripotentprogenitors.

EXAMPLE 3

Conditioned Medium Induces Hematopoiesis

Medium was conditioned by exposure to semi-confluent cultures of theimmortalized human stromal cell lines for one week. Both RPMI containing10% FCS and serum deprived (1% nutridoma-HU Boehringer Mannheim) mediawere used. The culture debris was pelleted by centrifugation at 2000×gfor 10 minutes and the supernatant was then aliquoted and frozen at -20°C. Conditioned media was thawed only once prior to use. Concentratedconditioned medium was made using Amicon centriprep 10 concentrator(Amicon, Beverly, Mass.) according to the manufacturers specificationsand protein content was determined using the BIO-RAD protein assay(Bio-Rad). Conditioned medium was assayed for colony stimulatingactivity in standard CFU assays and for cytokine content with ELISAsusing Quantikine kits (R & D Systems, Minneapolis, Minn.) according tomanufacturer's specifications. Supernatants were analyzed neat, at 1:2and at 1:5 dilutions.

The results showed that only conditioned media from HS-5 inducedproliferation of 38⁺ and 38^(lo) cells in the absence of stromal cells.FIG. 2A-C demonstrate the extent of proliferation of 38⁺ cells inducedby conditioned medium from HS-5 compared to conditioned medium fromHS-21 and a recombinant growth factor mix. Additional experiments usingconcentrated HS-5 CM indicated that a 12-15 fold expansion can beachieved within one week with a 4-6 fold increase in clonogenic cells.Additionally, methylcellulose assays were used to determine ifconditioned medium from HS-5, HS-21, HS-23, and HS-27 could supportcolony formation. Consistent with the shorter assay only conditionedmedium from HS-5 supported the growth of colonies from 38⁺ populationand the 38^(lo) population. FIG. 3 is a comparative analysis of theactivity of HS-5 conditioned medium, HS-21 conditioned medium (with andwithout serum), and growth factor mix (GF mix). HS-5 conditioned media,independent of serum content, generated an equivalent number of G/GMcolonies from 38^(lo) cells as the GF mix, however HS-5 conditionedmedium generated significantly more colonies from 38⁺ cells (FIG. 3A).In contrast, conditioned medium from HS-21 supported significantly fewerG/GM from both CD34⁺ subpopulations compared to HS-5 or GF mix. Therelative numbers of BFU-E generated by these conditioned media from38^(lo) cells were also significantly different and paralleled theobservations with the G/GM colonies (FIG. 3B). However, the GF mixgenerated significantly more BFU-E from 38⁺ cells than any conditionedmedium.

These results indicate that HS-5 and HS-21 secrete significant levels ofnumerous cytokines. However, only HS-5 can support CFU growth, whereasHS-21 conditioned medium did not support CFU even when concentrated8-fold. The possibility that HS-21 may contain an inhibitor was ruledout by mixing experiments.

The conditioned media from four cell lines were assayed for thecytokines G-CSF, GM-CSF, KL, LIF, IL-6, IL-1α, IL-3 and IL-11. Theresults indicated that only HS-5 and HS-21 conditioned medium containedsignificant amounts of these cytokines, and thus conditioned media fromthese lines were additionally tested for the presence of IL-1β, IL-IRA,IL-2, IL-7, IL-8, EGF, TNFα, TGFα and MIP-Iα. FIG. 4 demonstrates thatthe majority of these cytokines were present in HS-5 and HS-21supernatants at similar levels. HS-5 however, additionally secretesIL-1α, IL-1β, IL-1RA and LIF (at 0.4 to 1.8 ng/ml). Addition of thesecytokines to HS-21 supernatants did not support colony formation andneutralizing antibodies did not inhibit HS-5 supernatants. Thus, HS-5was secreting a cytokine that could support colony formation by itselfor in combination with other factors. To identify genes expressed inHS-5 and not in HS-21, a differential display technique was used,identifying two isolated bands which were uniquely expressed by HS-5.IL-3 was not found in any supernatant and was not detectable using rtPCRfor IL-3 message in mRNA from the HS-5 or HS-21 cell lines.

Together, these observations suggest that the factors responsible fordifferentiation and proliferation of committed progenitors are distinctfrom those required for maintenance of the immature pre-CFC pool.Moreover, it suggests that these maintenance factors are extracellularmatrix or membrane associated.

All publications, patents and foreign patent publications are hereinincorporated by reference to the same extent as if each individualpublication, patent or patent publication was specifically andindividually indicated to be incorporated herein by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

MICROORGANISM DEPOSIT INFORMATION

A deposit of the human stromal cell line HS-5 was made on May 5, 1995 onbehalf of Fred Hutchinson Cancer Research Center with the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209under the terms of the Budapest Treaty and designated accession numberATCC CRL 11882.

What is claimed is:
 1. An immortalized human stromal cell line, which isHS-5 as deposited with the American Type Culture Collection anddesignated accession number ATCC CRL
 11882. 2. A subclone of theimmortalized human stromal cell line of claim
 1. 3. The immortalizedhuman stromal cell line of claim 1, which has been irradiated.
 4. Theimmortalized human stromal cell line of claim 3 in a combined in vitroculture with human hematopoietic precursor cells.
 5. The immortalizedcell line of claim 1 in a combined in vitro culture with humanhematopoietic precursor cells.